Immunogenic composition forming a vaccine, and a method for its manufacture

ABSTRACT

An immunogenic composition forming a vaccine includes a nanoparticle adjuvant comprising at least a nanoparticle, wherein the at least a nanoparticle comprises a lipid layer exterior including a plurality of lipids, cholesterol, and a primary alkyl amine including a positively charged amino group head and at least a carbon tail and an antigen incorporated in the at least a nanoparticle, wherein the antigen comprises a spike protein from a coronavirus.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of U.S. ProvisionalPatent Application Ser. No. 63/003,254, filed on Mar. 31, 2020 andentitled “LIPOSOMAL VACCINE ADJUVANT FOR VIRUS SPIKE PROTEINS ANDMETHODS OF MAKING AND USING SAME,” the entirety of which is incorporatedherein by reference.

FIELD OF THE INVENTION

The present invention generally relates to the field of vaccinecompositions and methods of making and using the same. In particular,the present invention is directed to an immunogenic composition forminga vaccine, and a method for its manufacture.

BACKGROUND

Coronaviruses are an emerging pandemic threat that humans rarely haveinnate immunity to. Infection typically results in mild respiratorysymptoms but can be more serious in infants and older adults, especiallythose with underlying comorbidities. Respiratory infection is secondonly to malaria as a cause of infant mortality worldwide and accountsfor substantial hospitalization burden in both age groups in developedcountries. Moreover, some pathogens, such as newly emergent zoonoticviral strains, can pose a significant risk of mortality to the generalpopulation as well. Despite intensive effort, including numerous vaccinecandidates currently in preclinical or clinical development, a safe andeffective vaccine for coronaviral infections is still an elusive goal.

SUMMARY OF THE DISCLOSURE

In an aspect, an immunogenic composition forming a vaccine includes ananoparticle adjuvant comprising at least a nanoparticle, wherein the atleast a nanoparticle comprises a lipid layer exterior including aplurality of lipids, cholesterol, and a primary alkyl amine including apositively charged amino group head and at least a carbon tail and anantigen incorporated in the at least a nanoparticle, wherein the antigencomprises a spike protein from a coronavirus.

In another aspect, a method of manufacturing an immunogenic compositionforming a vaccine includes forming a nanoparticle adjuvant, wherein theadjuvant comprises a plurality of nanoparticles and each nanoparticlecomprises a lipid layer exterior including a plurality of lipids,cholesterol, and a primary alkyl amine including a positively chargedamino group head and at least a carbon tail. The method includesproviding an antigen, the antigen comprising a plurality of spikeproteins from a coronavirus. The method includes combining the antigenwith the nanoparticle adjuvant.

These and other aspects and features of non-limiting embodiments of thepresent invention will become apparent to those skilled in the art uponreview of the following description of specific non-limiting embodimentsof the invention in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of illustrating the invention, the drawings show aspectsof one or more embodiments of the invention. However, it should beunderstood that the present invention is not limited to the precisearrangements and instrumentalities shown in the drawings, wherein:

FIG. 1 is a schematic diagram of an exemplary embodiment of animmunogenic composition;

FIG. 2 is a schematic diagram of an exemplary embodiment of animmunogenic composition;

FIG. 3 is a schematic diagram of an exemplary embodiment of animmunogenic composition;

FIG. 4 is a schematic diagram of an exemplary embodiment of animmunogenic composition;

FIG. 5 is a flow diagram illustrating an exemplary embodiment of amethod for manufacture of an immunogenic composition;

FIG. 6 is a flow diagram illustrating an exemplary embodiment of amethod for manufacture of an immunogenic composition;

FIG. 7 is a bar graph illustrating experimental results describingrelative immunogenicity;

FIG. 8 is a bar graph illustrating experimental results describingrelative immunogenicity;

FIG. 9 is a bar graph illustrating experimental results describingrelative immunogenicity;

FIG. 10 is a bar graph illustrating experimental results describingrelative immunogenicity;

FIG. 11 is a bar graph illustrating experimental results describingstability over time; and

FIGS. 12 and 13 are histograms illustrating experimental resultsdescribing stability over time. The drawings are not necessarily toscale and may be illustrated by phantom lines, diagrammaticrepresentations, and fragmentary views. In certain instances, detailsthat are not necessary for an understanding of the embodiments or thatrender other details difficult to perceive may have been omitted.

DETAILED DESCRIPTION

Embodiments disclosed herein present a novel vaccine designed againstspike proteins from coronaviruses, such as SARS-CoV-2, using alipid-based nanoparticle formulation. Formulation may include aliposomal formulation. A resulting vaccine may be scalable, flexible inits antigen presentation, and have the potential for stability outsidethe cold chain. In an embodiment, a vaccine may include a positivelycharged chemical vaccine additive for cell targeting, and may include aliposomal vaccine adjuvant with entrapped, embedded, and/or surfaceadsorbed viral spike proteins and protein complexes of a variety ofviruses belonging to the Coironaviridae family of viruses for efficientpresentation of the viral spike proteins to the immune system. Thispresentation of the viral spike protein antigen may induce a strongimmune response in vivo and lead to the generation ofcoronavirus-neutralizing antibodies and significant amelioration ofinfection to coronaviral infections.

Embodiments may include, as a non-limiting example, a liposomal or otherlipid-based nanoparticle vaccine formulation that includes entrapped,embedded, and/or surface adsorbed glycoproteins, of a variety ofoligomeric states, found on enveloped viruses such as coronaviruses.“Spike proteins” are glycoproteins responsible for binding to host cellsurface receptors and subsequent viral entry and represent a preeminentsource of potential antibody-recognizing antigens. These spike proteincomplexes are believed to elicit a protective adaptive immune responsein generating neutralizing antibodies against the viral surface,resulting in antibody opsonization and prevention of viral-mediatedentry into host cells via spike protein interactions with host cellreceptors. A potential avenue to combat such viruses may thus be tocreate a vaccine against these spike proteins, and other similarglycoproteins, which have been extensively characterized for other humancoronavirus such as SARS and MERS, as well as non-human animalcoronaviruses such as PEDV, FPIV, and MHV. Presentation of theseantigenic glycoproteins in a more physiologically relevant,lipid-associated presentation to the immune cells may be essential toeliciting an appropriate immune response.

The pandemic caused by the Severe Acute Respiratory Syndrome Coronavirus2 (SARS-CoV-2), previously known as the 2019 novel coronavirus(2019-nCoV) is an example of such an enveloped virus that has a trimericspike (S) protein at its viral surface. The trimeric S protein ofSARS-CoV-2, consisting of an S1 protein and a S2 protein, is responsiblefor binding to the host cell surface receptor, Angiotensin-convertingenzyme 2 (ACE2), and trigger subsequent receptor-mediated viral entryinto the host cell. Symptoms in infected patients include fever,coughing, malaise, night sweats, headache, and breathing difficultiesthat may ultimately be fatal, especially in elderly patients or thosewith underlying diseases. Currently, there are no effective vaccinesagainst human coronaviruses. Additionally, there are human and non-humancoronaviruses with significant health and/or economic impact orpotential impact including, without limitation, SARS (Severe AcuteRespiratory Syndrome), MERS (Middle Eastern Respiratory Syndrome), MHV(Mouse Hepatitis Virus), PEDV (Porcine Epidemic Diarrheal Virus), andFIPV (Feline Infectious Peritonitis Virus); the last three infect onlynon-human animals, but boast high mortality rates and rates of infectionand attack economically and scientifically important species.

Referring now to FIG. 1, an exemplary embodiment of an immunogeniccomposition 100 is illustrated. Immunogenic composition 100 includes ananoparticle adjuvant. An “adjuvant,” as used in this disclosure, is apharmacological and/or immunological agent that improves, or helps tostimulate, an immune response of a vaccine, antigen, or otherimmunologically active compound. Nanoparticle adjuvant includes at leasta nanoparticle 104. A “nanoparticle,” as used in this disclosure, is aparticle of matter between 1 and 2000 nanometers in diameter. Forinstance, and without limitation, at least a nanoparticle 104 may beengineered to have an average size less than 500 nm in diameter. Atleast a nanoparticle 104 may be engineered to have a diameter between 50nanometers and 2000 nanometers. At least a nanoparticle may have adiameter between 50 nanometers and 1000 nanometers. At least ananoparticle may have an average diameter of approximately 200-300nanometers. At least a nanoparticle may include a plurality ofparticles, a large majority of which are between 80 nanometers and 500nanometers in diameter; a small number of outliers may be between 5nanometers and 1200 nanometers. At least a nanoparticle 104 may includea plurality of nanoparticles, which may be suspended, withoutlimitation, in an aqueous medium, lyophilized, and/or cryogenicallypreserved as described in further detail below. At least a nanoparticle104 includes a lipid layer 108 exterior including a plurality of lipids,which may vary in physicochemical properties. Lipid layer 108 exteriormay include, without limitation, a lipid monolayer, bilayer, and/ormulti-lamellar construction and/or lipid corona about a non-liposomenanoparticle, which may include any nanoparticle as described above, orthe like. At least a nanoparticle 104 may include, for instance, aliposome. A “liposome,” as used in this disclosure, is a vesicleenclosed by a lipid and/or phospholipid bilayer. Alternatively oradditionally, at least a nanoparticle 104 may include a micelle, definedas a lipid monolayer enclosure, a bicelle, an amphipol, a nanodisc, astyrene-maleic acid lipid particle (SMALP), and/or a nanostructure suchas a piece of inorganic and/or organic material such as metal-based,metal oxide, carbon-based, immune-stimulating complex (ISCOM), proteincages, or other nanoparticles with a lipid material, or the like. Lipidand/or lipids making up lipid layer 108 and/or nanoparticle 104construction may include, without limitation, phospholipids such asdipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylcholine(DOPC), non-phospholipid lipids incorporating and/or combined withpolyethylene glycols (PEGs), such as without limitation, PEGylatedlipids, PEG-conjugated lipids, or the like, zwitterionic, neutral,cationic, and/or anionic phospholipids and non-phospholipids such asphosphatidylcholine, ceramides, phosphatidylethanolamine, saturated,monounsaturated and/or polyunsaturated fatty acid lipids, and the like.Lipids may be selected from the FDA GRAS list for approved excipients,for instance to guard against any biosafety issues. Lipid layer 108includes cholesterol 112 and/or cholesterol 112 derivatives, includingcholesterol with other functional groups added on; cholesterolderivatives may include, without limitation, cholesterol derivativesdenoted as disterol-phospholipid Bis-Azo-PC, Chol-T, Chol-Q, or thelike. Lipid layer 108 includes a primary alkyl amine 116, defined as astructure having an amine functional group and one or more carbon tailsin an unbranched and/or branched carbon chains formation; primary alkylamine 116 may include without limitation nonadecanamine, stearylamine,heptadecylamine, cetylamine, tripentyalmine, and/or isomers of the alkylamines, or the like.

Primary alkyl amine 116 includes a positively charged amino group headand at least a carbon tail. Non-limiting examples of primary alkyl amine116 include stearylamine (SA), pentylamine (C₅H_(13N)), alkyl amines ofany carbon length, as well as branched alkyl amines such astripentyalmine, amylamines, or the like, mixtures of isomers of theabove, and/or alkyl amines with varying degrees of poly- andmono-unsaturated carbon chains, such as alkene and/or alkyne substitutedalkyl amines. Primary alkyl amine 116 may be positively charged. As aresult, lipid layer 108 and/or at least a nanoparticle 104 may bepositively charged; in an embodiment, positive charge of primary alkylamine 116 may neutralize a net negative charge of at least ananoparticle 104 and/or may cause overall charge of at least ananoparticle 104 to become positive. In an embodiment, and withoutlimitation, where at least a nanoparticle 104 is positively charged, atleast a nanoparticle 104 may attract spike proteins having negativecharges, improving entrapment and/or adsorption to lipid surface ofspike protein. In a non-limiting example, a positive charge of combinednanoparticle and antigen may further have an effect of attraction tonegatively charged cell membranes of immune and/or somatic cells, whichmay cause combined nanoparticle and antigen to contact and/or deliverinto such cells the antigens; this may increase immunogenic effect ofthe resulting vaccine by improving cell-targeting. In some embodiments,spike proteins may alternatively or additionally complex bind to lipidlayer; for in instance, spike protein may interact and change proteinconformation to effect a complex bind, which may occur as a non-limitingexample where a formulated vaccine is lyophilized and thenreconstituted. In some embodiments, where antigen has a positive charge,alkyl amine and/or an additional compound having a negative charge, suchas without limitation DPPG (dipatmityl, dioleoyl,disterylphosphatidylglycerol), alginate, and/or polyalginate, may beused to give lipid layer a net negative charge. Generally, where antigenhas an electric charge with a first polarity, lipid layer exterior mayhave an electric charge with a second polarity, wherein the firstpolarity differs from the second polarity; i.e. a where the firstpolarity is negative the second polarity may be positive, andvice-versa.

Still referring to FIG. 1, as a non-limiting example, materials used inlipid layer 108 and/or liposome may include cholesterol 112 atapproximately 20 mol %, saturated lipids DPPC in an amount ofapproximately 40 mol %, SA, positively charged, at approximately 15 mol%, and unsaturated lipid DOPC neutral, at approximately 25 mol %. In anon-limiting, illustrative embodiment, ratios of lipids may be in arange of DPPC:DOPC:cholesterol 112:alkyl amine molar ratio is40:20-30:20:10-30. In an embodiment, differing molar ratios may be usedto optimize various recombinant forms of spike proteins, and/or improveadsorption of coronavirus spike proteins from other species.

Further referring to FIG. 1, immunogenic composition 100 includes anantigen incorporated in the at least a nanoparticle 104. An “antigen,”as used in this disclosure, is a viral molecule and/or molecularstructure that may induce an antigen-specific antibody response and/orresult in immune cell antigen receptor-binding. In an embodiment,antigen includes a spike protein from a coronavirus, which may includeany virus in the subfamily Orthocoronavirinae. A “spike protein,” asused in this description, is a protein and/or glycoprotein structurethat projects from, lies, on, or traverses a surface of a virusparticle. A spike protein in a coronavirus may be referred to as an “S”protein, for instance S1 or S2. Spike protein may include withoutlimitation a trimeric protein complex or one or more subunits thereof,such as an S1 subunit, an S2 subunit, or the like, or homo- and/orhetero-oligomeric forms of these proteins. In an embodiment, and asdescribed in further detail below, an S2 subunit may be embedded in alipid bilayer of a virus particle, while a corresponding S1 subunit maybind to the S2 protein and project beyond the bilayer, extending awayfrom the virus particle surface to engage host cells; this may enable acoronavirus to penetrate such cells by binding, for instance, the humanACE2 receptor, leading to internalization of the virus particle and/or apayload thereof, and ultimately infection. Spike protein, and/or anysub-unit thereof as described above may contain at least apost-translational modification (PTM) such as glycosylation,phosphorylation, acetylation, ubiquitination, isoprenoid attachment, orthe like. Spike protein may be recombinant, and/or may be harvested frompartial and/or whole viral particles. For instance, and withoutlimitation, spike protein may include NCP-CoV (2019-nCoV) spike protein(S1+S2 ECD) and/or SARS-CoV-2 (2019-n-Cov) Spike S1-His recombinantprotein. Spike protein may include a His tag; in such an example, a ‘Histag’ may be a poly-histidine amino acid fusion tag, as part of arecombinant spike protein, used for purification of the recombinantspike protein. Recombinant spike protein forms may contain purificationtags, artifacts, or the like, including histidine tags, maltose-bindingprotein (MBP) tags, streptavidin-biotin tags, FLAG tags, and the like.Recombinant spike proteins and/or any viral glycoproteins used innanoparticle formulations may originate from prokaryotic and/oreukaryotic recombinant expression systems, for instance and withoutlimitation, mammalian cell expression, bacterial cells expression, yeastcell expression, and insect cell-baculoviral expression systems, and thelike. Recombinant spike proteins and/or viral glycoproteins may bemodified in DNA sequence to optimize recombinant expression and/orpurification, but still result in faithfully recapitulated amino acidsequences resembling native viral proteins. Spike protein may beHPLC-verified. Persons skilled in the art, after reviewing thedisclosure in its entirety, will be aware of the various forms purifiedrecombinant viral proteins may present.

With continued reference to FIG. 1, a spike protein or other antigen mayinclude, without limitation, a glycoprotein. A glycoprotein is asurface-exposed viral structural protein that containsglycans—carbohydrate PTMs on the surface and/or within the protein. AnS1 glycoprotein of a coronavirus may be, without limitation ahomotrimer, a monomer, and/or a dimer. A process whereby glycans arechemically modified onto a surface of a glycoprotein is referred to asthe process of “glycosylation,” and is a post-translational modification(PTM) defined as a chemical attachment to a protein after synthesis inthe cell. Glycosylation may function to shield, or otherwise alter,antigenic sites on a virus for immune cell avoidance. Differentglycosylation states may exist for glycoproteins such as SARS-CoV-2glycoproteins, including without limitation other PTMs such ashydroxylation, methylation, lipidation, acetylation, disulfide bondformation, ubiquitination, SUMOylation, phosphorylation, proteolysis,and the like, as described above. Depending on the recombinant source,there may be final glycosylation states that differ in theirmodification pattern, amount, branching, and physicochemical properties,and potentially their immunogenicity; for instance, different forms ofglycosylation may result from recombinant production of spike proteinsin insect, mammalian, bacterial, and yeast cells or other organisms usedfor recombinant manufacture of the spike protein. In some embodiments,spike proteins used may evince varying truncated and/or mutated formssuch as forms having various amino acid mutations.

Further referring to FIG. 1, in alternative embodiments, antigen mayinclude one or more surface proteins of other types of viruses, such aswithout limitation influenza virus or respiratory syncytial virus (RSV).Antigen may alternatively or additionally include surface proteinsbesides spike proteins, such as “M” proteins; in an embodiment, use of amixture of S proteins and M proteins may modify and/or improve overallimmunogenicity, stability, glycoprotein packing, or the like.

Still referring to FIG. 1, antigen is incorporated in the at least ananoparticle 104. “Incorporation,” as used herein, is any form ofattachment, adsorption, and/or entrapment on or in a nanoparticle; forinstance, and without limitation, antigen may be adsorbed to a surfaceof lipid layer 108. As a non-limiting example, and as shown in FIG. 2,spike protein may include an S1 protein 200 without an S2 in complexwith it, which may be attached to and/or adsorbed to lipid layer. As afurther non-limiting example, and as illustrated in FIG. 3, spikeprotein may include an S2 protein 300 embedded in lipid layer 108,adsorbed to lipid layer 108 and/or bilayer, and/or interacting withlipid layer 108 and/or bilayer, and an S1 protein 304 projecting fromthe lipid layer 108. As a further non-limiting example, and as shown inFIG. 4, where nanoparticle includes or is a liposome, spike protein maybe entrapped in an aqueous compartment of the liposome, and/or may beadsorbed to lipid layer as well. Incorporation may include entrapmentbetween layers of a bilayer; for instance, where lipid layer 108includes a bilayer and/or multi-lamellar construction, spike protein maybe entrapped within the bilayer.

With continued reference to FIG. 1, incorporation may be achieved byoptimizing, or otherwise altering, the lipid composition, surface chargeof the liposome, and size of the liposomes, as well as any otherphysicochemical property. For example, a spike protein such as an S1S2spike protein of SARS-CoV-2 may be a negatively charged protein, forinstance with acidic patches, that binds more efficiently to positivelycharged lipid surfaces and/or liposomes through favorable ionicinteractions. Therefore, mixing a positively charged nanoparticle suchas a positively charged liposome with an S1S2 spike protein ofSARS-CoV-2 may result in protein adsorption to the liposome and/ornanoparticle surface as well as some entrapment inside the liposomeand/or nanoparticle. This particle-protein complex may subsequentlyinteract with the immune cells and elicit a protective immune responsein generating antibodies to the S1S2 spike protein. Such a protocol maybe used for other antigenic proteins in generating a liposomal vaccine.Adsorption may be achieved, without limitation through ionic,hydrophobic, Van der Waals interactions, hydrogen bonding, and/orthrough covalent interactions and/or conjugation. Methods of manufactureas described in further detail below may entrap the target antigeninside a liposome as well as decorating the liposome surface with spikeproteins by adsorption through molecular interactions. Where at least ananoparticle 104 includes a liposome, liposome composition may bechemically modified to an appropriate surface charge that maximizesbinding of target antigen to surface of the liposome and forpresentation of the liposomes to the immune cells.

Further referring to FIG. 4, immunogenic composition may bemanufactured, stored, and/or prepared in one or more lyophilized formsand/or in one or more dried states using various drying technologiessuch as without limitation spray drying, vacuum drying, foam drying, orthe like. For instance, and without limitation, immunogenic compositionand/or one or more components thereof may be presented in an on-demandformat in which composition is lyophilized for stability, thenreconstituted for use. For instance, and without limitation, immunogeniccomposition may be formulated as a lyophilized composition, afterincorporation of antigen in at least a nanoparticle 104. Alternativelyor additionally, nanoparticle adjuvant may be lyophilized separately andreconstituted with the antigen; in other words, incorporation may beperformed concurrently with reconstitution. Reconstitution may refer toresuspension, hydration, solvation, or otherwise reconstituted inaqueous solution, including buffer compositions such as phospho-bufferedsaline (PBS), or the like. In further non-limiting illustrativeembodiments, reconstitution of a lyophilized nanoparticle, such as aliposome-glycoprotein complex, may be performed with varying saltconcentrations, such as sodium chloride. In an embodiment,reconstitution of separately lyophilized nanoparticles with antigen maycause antigen to be trapped within a vesicle and/or other interior suchas an aqueous interior of a liposome as well as attached to a surfacethereof.

Still referring to FIG. 4, immunogenic composition 100 may include atleast one lyoprotectant. A lyoprotectant, as used in this disclosure, isa substance that protects a substance during cryogenic freezing, duringfreeze-drying, and/or during freeze-thaw cycles. At least onelyoprotectant may include, without limitation, a polyol, such as withoutlimitation sucrose, trehalose, mannitol, or the like, and/or at leastone ionic strength balancing component, including for instance a salt,pH buffer, or the like. At least one lyoprotectant may include an aminoacid, such as without limitation glycine, arginine, or the like. Personsskilled in the art, upon reviewing the entirety of this disclosure, willbe aware of various alternative or additional lyoprotectants,cryoprotectants, and the like that may be employed consistently withthis disclosure.

Still referring to FIG. 4, immunogenic composition 100 may include anysuitable combination of elements including without limitation any set offormulations as set forth below in table 1. Formulations may includewithout limitation protectants such as sugar, pH control buffers,preservatives such as polysorbate 20%, and/or an ingredient such as NaCLor other salts to balance ionic strength.

TABLE 1 Exemplary Formulations Polysorbate Vaccine S1 S1-S2 Lipid^(a) pHBuffer 20% Sugar B-S1 10 μg/mL — 25 mg/mL 7.2 Histidine 0.05 10% SucroseB-S1S2 — 10 μg/mL 25 mg/mL 7.2 Histidine 0.05 10% Sucrose ^(a)includingcholesterol 112 and alkyl amine.

Still referring to FIG. 4, vaccine may be administered in any suitablemanner. In an embodiment, vaccine may be injectable. Vaccine mayalternatively or additionally be absorbed through a mucous membrane, forinstance via aerosolized delivery to the nostrils and/or lungs.Alternatively or additionally, vaccine may be administered using apatch, such as without limitation a microneedle patch that deliverslyophilized vaccine in powder form; as a non-limiting example,lyophilized vaccine may be included in soluble microneedles which uponinsertion to tissue of a living organism may dissolve in fluids thereof,reconstituting and activating the vaccine. As a further non-limitingexample, lyophilized vaccine may be delivered in an implant such as asoluble or insoluble needle inserted under the skin or into other tissueallowing fluids of the subject tissue to reconstitute and disseminatethe vaccine. Vaccine may be delivered in liquid and/or lyophilized formto any mucous membrane; for instance and without limitation, vaccine maybe delivered as a lyophilized inhalable powder for absorption in nasaland/or pulmonary surfaces. Vaccine may be delivered orally, for instancein a needle or other device for injecting lyophilized vaccine intoand/or across digestive tissues, which may be delivered in a capsuledesigned to disintegrate in one or more digestive juices. Vaccine inlyophilized form may be delivered by a nanobot.

Referring now to FIG. 5, an exemplary embodiment of a method 500 ofmanufacturing an immunogenic composition forming a vaccine isillustrated. At step 505, a nanoparticle adjuvant is formed.Nanoparticle adjuvant may include any nanoparticle adjuvant as describedabove. Nanoparticle adjuvant includes a plurality of nanoparticles,which may include any nanoparticles as described above. Eachnanoparticle includes a lipid layer 108 exterior including a pluralityof lipids, cholesterol 112, and a primary alkyl amine 116 including apositively charged amino group head and at least a carbon tail, forinstance and without limitation as described above. Lipid layer 108 maybe positively charged, for instance by application of a concentration ofa positively charged alkyl amine as described above. Each nanoparticlemay include, without limitation, a liposome.

Still referring to FIG. 5, formation of nanoparticle adjuvant mayinclude formation of a suspension of liposomes. Formation may includehydrating a dried lipid blend, such as without limitation a freeze-driedlipid blend, and extruding the resulting solution through a filterhaving pore sizes at approximately an upper limit of a desired liposomediameter, which may be a desired diameter falling into ranges and/oraverage sizes as described above.

At optional step 510, combining may include lyophilizing thenanoparticle adjuvant, for instance and without limitation as describedin further detail below.

At step 515, and still referring to FIG. 5, method 500 includesproviding an antigen. Antigen includes a plurality of spike proteinsfrom a coronavirus, as described above. For instance, and withoutlimitation, spike protein may include an S1 protein. Spike protein mayinclude an S1S2 protein. Intact spike protein and/or various specificdomains, such as S1 and S2 subunits may be recombinant; for instance,and without limitation, intact spike protein and/or specific domainsand/or subunits may be manufactured using mammalian cell-culture basedexpression systems, or a plurality of expression systems as describedabove. Alternatively or additionally, whole and/or partial virusparticles may be generated and/or replicated, and spike proteins and/orsubunits may be extracted, separated from, sheared off, or otherwisepurified from such whole or partial virus particles, or virus-likeparticles.

With continued reference to FIG. 5, spike protein may include aglycoprotein. Glycosylation of spike protein may occur during productionand/or replication of virus particles. Glycosylation may be variedacross batches and/or populations of spike proteins or among individualspike proteins; this may generate a recombinant glycoprotein librarywith glycoproteins of varying degrees of glycosylation. In anembodiment, spike proteins of varying glycosylation may be combined inthe antigen; this may result in nanoparticles, such as liposomes,incorporating a plurality of different glycoproteins of the samespecies. In an embodiment, such liposomes may allow for greaterimmunogenicity. For instance, and without limitation, preparation of S1may include reconstituting S1 in water for injection (WFI) to generate agiven concentration, including without limitation a 250 μg/mL S1 stocksolution. Specific amounts of S1 stock solution may then be diluted inspecific amounts of a formulation buffer, which may include withoutlimitation a 0.01% polysorbate 20/sucrose/histidine buffer to aconcentration of approximately 10 μg/mL S1. As a further non-limitingexample, a 550 μg/mL S1S2 stock solution, which may be reconstitutedwithout limitation as described above, may be diluted in specific amountof formulation buffer, including any buffer as described above, to aconcentration of approximately 10 g/mL S1S2. Buffer may generallyinclude any buffer offering a buffering capacity within a pH range of6.0-7.5, including without limitation histidine and/or phosphate buffer.Buffer may include a polysorbate 20 or 80 concentration within a rangeof 0.001%-0.05%. Buffer may include a polysorbate 20 or 80 concentrationwithin a range of 0.001%-0.05%.

At step 520, and still referring to FIG. 5, antigen is combined withnanoparticle adjuvant. In an embodiment, a suspension of protein antigenmay be added to an aqueous suspension of nanoparticle adjuvant, using amixing device to get a homogeneously distributed antigen-liposomemixture. Mixing device may include, without limitation, a magneticstirrer, a sonication device, a homogenizer, or the like. Mixture mayalternatively or additionally be swirled mechanically or manually.Combination according to this technique may tend to producesurface-mounted spike proteins and/or spike proteins adsorbed to lipidsurface, for instance as described above. Where nanoparticles have acharge with an opposite polarity to a charge of antigen, antigen mayadsorb to the liposomes rapidly. In an embodiment, lyophilizednanoparticle adjuvant may be reconstituted using a suspension of antigenin a buffer solution.

Alternatively or additionally, and continuing to refer to FIG. 5,hydration of lipid blend prior to extrusion may be performed with asolution and/or suspension of antigens; in other words, generation ofnanoparticle adjuvant may be performed concurrently with and/orsubsequently to combination of antigen with nanoparticle adjuvant. Thismay produce liposomes that include both entrapped and adsorbed antigens.

At step 525, and still referring to FIG. 5, vaccine particles formedaccording to any process as described above may be lyophilized.Lyophilized vaccine particles may be delivered and/or stored inlyophilized form and may subsequently be reconstituted prior toadministration and/or may be administered in powdered and/or lyophilizedform as described above.

Referring now to FIG. 6, a flow diagram illustrating an exemplaryembodiment of a method 600 of manufacturing an immunogenic compositionforming a vaccine is illustrated. At step 605, an antigen is provided;antigen may include, without limitation, any antigen described above,including without limitation spike proteins and/or glycoproteins of acoronavirus, and/or portions thereof. For instance, and withoutlimitation, antigen may include S1 glycoproteins and/or S1S2glycoproteins. Provision of antigen may be performed, withoutlimitation, according to any process described above in reference toFIG. 5.

At step 610, and still referring to FIG. 6, a dry lipid blend may beprovided and/or formed. As a non-limiting example, a blend of DPPC,DOPC, cholesterol and stearylamine (40:25:20:15:mol %, respectively maybe dissolved in a chloroform/methanol/water solution. Solution may bedried, for instance in a rotary evaporator under a stream of nitrogengas. Solution may subsequently be dissolved in a co-solvent ofcyclohexane/80% tertiary-Butyl alcohol (v/v) at a final lipidconcentration of 20 mg/ml, for instance in aliquots of 50 mg oflipid/vial. Vials may then be lyophilized to obtain a dried lipid blend;vials may be sealed with nitrogen (N2) gas prior to partial placement ofa stopper. Vials may then be freeze dried under a blanket of N2 gas, forinstance in a freeze-dryer. As a non-limiting example lipid-blend may belyophilized by freezing at −45° C., primary drying at −30 to −35 C, andsecondary drying at 25-30° C.

Further referring to FIG. 6, lipid blend may be hydrated with an antigensolution and/or suspension, as illustrated at step 615. Antigen solutionmay include, without limitation antigen combined with a buffer to form asuspension. Buffer may include, without limitation, a lyoprotectant,which may include any lyoprotectant described above. As a non-limitingexample, a 200 gr 10 mM histidine, 10% sucrose buffer may be prepared. ApH of buffer may be adjusted to approximately 7.2 when measured at 25degrees Celsius. Buffer may be sterile filtered through a filter such aswithout limitation a 0.2 μm or 0.22 μm filter. Buffer may then becombined with the antigen mixture; alternatively or additionally,combination with antigen may occur concurrently with or subsequent toreconstitution of lyophilized nanoparticles with buffer. For instance,and without limitation, lyophilized lipid-blend may be hydrated withfiltered antigen buffer solution and vortexed and/or sonicated untillipids are hydrated and liposomes are formed. Hydration with antigensolution may form a colloidal vaccine solution. At step 620, colloidalvaccine solution may be extruded, for instance using filtration asdescribed above in reference to FIG. 5, to form desired particle sizes.As a non-limiting example, where positively charged dried lipid-blendsas described above, may be hydrated with a specific amount of acorresponding spike protein solution such as without limitation a 40μg/mL spike protein solution; pH of spike protein solution may match pHof lipid and/or nanoparticle solution. A resulting combined solution maybe extruded through filters; for instance, a vaccine particle solutionmay be extruded through a membrane filter, such as through 2×400 nmmembrane filters in an extruder such as a 10 mL extruder. As a furthernon-limiting example, solution may be extruded ten times through two 400nm polycarbonate filters in a 10 ml extruder at 50-100 psi usingnitrogen gas. Extrusion may be performed gradually, for instance in alaminar flow hood using N2 gas. This procedure may be repeated one ormore times; extrusion may be repeated until all solution has passedthrough the extruder 10 times. A resulting solution may be dispensed invials; for instance, solution may be dispensed in 3 mL depydrogenatedglass vials, for instance filling 800 μL fill volume. Dispensation maybe performed in a laminar flow hood. Dispensation may be performed usinga fine 1 mL pipette and sterile disposable pipette tips. Preparationaccording to steps 615 and 620 may be referred to herein as formulation“C”; for instance, where antigen is a solution of S1 spike proteins,formulation may be referred to as CS1, while where antigen is a solutionof S1S2 spike proteins, formulation may be referred to as CS1S2.

Alternatively or additionally, and still referring to FIG. 6, at step625 dried lipid blend may be hydrated with formulation buffer, forinstance and without limitation as described above, without antigen toform nanoparticle adjuvant alone as a colloidal solution. For instance,and without limitation, lyophilized lipid-blend may be hydrated withfiltered buffer, vortexed and/or sonicated until lipids are hydrated andliposomes are formed. Nanoparticle adjuvant may be extruded and/ordispensed in vials as described above, as illustrated at step 630.

In some embodiments, and with continued reference to FIG. 6,nanoparticle adjuvant as formed at steps 625 and 630 may be combined inits form as a colloidal solution with antigen, for instance by mixing aprotein solution of antigen with the colloidal solution ofnanoparticles, as illustrated at step 635; this may be implemented,without limitation, as described above in reference to FIG. 5. Aformulation as described in reference to steps 625, 630, and 635 isreferred to herein as formulation “A”; for instance, where antigen is asolution of S1 spike proteins, formulation may be referred to as AS1,while where antigen is a solution of S1S2 spike proteins, formulationmay be referred to as AS1S2.

Alternatively or additionally, and still referring to FIG. 6,nanoparticle adjuvant may be lyophilized, as illustrated at step 640. Atstep 645, lyophilized nanoparticle adjuvant may be reconstituted withantigen, for instance and without limitation using antigen in a bufferedsolution. A formulation as described in reference to steps 625, 640, and645 is referred to herein as formulation “B”; for instance, whereantigen is a solution of S1 spike proteins, formulation may be referredto as BS1, while where antigen is a solution of S1S2 spike proteins,formulation may be referred to as BS1S2. In an embodiment,reconstitution of freeze-dried nanoparticles (“B”) with spike proteinantigens to be trapped within a vesicle such as an aqueous interior of aliposome, as well as adsorbed to and/or trapped in lipid layer 108, forinstance as illustrated above in reference to FIG. 4. In an embodiment,mixture of lyophilized adjuvant with antigen may be performed shortlybefore administration; in other words, lyophilized adjuvant and antigenmay be transported and/or stored separately and combined at or near asite of administration.

At step 650, and still referring to FIG. 6, any vaccine formulationdescribed above may be lyophilized. Lyophilized vaccines may be denotedas formulation “D”; for instance, where antigen is a solution of S1spike proteins, formulation may be referred to as DS1, while whereantigen is a solution of S1S2 spike proteins, formulation may bereferred to as DS1S2. At least one lyoprotectant as described above maybe included with combination of antigen with nanoparticle adjuvant.Lyophilization and/or inclusion of lyoprotectants may be accomplished inany manner consistent with descriptions provided above. For instance,and without limitation vaccines may be filled in vials and freeze-driedin a freeze-drier such as a Vertis Genesis 12XL by first freezing thesolution to −45° C. at 0.5° C./min, followed by a 2-hour hold. Furthercontinuing the example, primary drying may be performed below theprimary glass transition of the frozen solution (Tg′), for example at−35° C. shelf temperature for at least 10 hours at a chamber pressure of100 mTorr or until completion of primary drying. After primary drying,and still continuing the example, a shelf may be ramped up to 25° C. at0.2° C./min. Still continuing the example, secondary drying may beperformed at 25° C. shelf temperature for 4 hours at a chamber pressureof 100 mTorr. Second lyophilization of combined proteins and liposomesmay cause a complex interaction between antigen, such as S1 or S1S2, andlipids and/or sugar and/or other lyoprotectant. At step 655,freeze-dried vaccines, such as freeze-dried S1 and S1S2 liposomalvaccines (referred to here as D-S1 and D-S1S2) may be reconstituted withwater for injection (WFI). A resulting liposome solution may include a25 mg/mL liposome (or between 1 and 50 mg/mL) and 10 ug/mL S1 or S1S2.Alternatively, lyophilized vaccine may be directly administered.

At step 660, vaccine may be administered, according to any suitableprocess for administration, including without limitation any processdescribed in this disclosure. The above-described methods are providedfor exemplary purposes only; any combination of method steps asdescribed in this disclosure is considered within the scope of thisdisclosure.

Reference is now made to immunization study results regarding study ofimmunization to coronavirus in mice. Study was conducted according to anapproved Animal Care and Use Protocol (ACUP). 2×50 μl of eachformulation tested was injected intramuscularly (im) in the leg of fivefemale BALB-C mice on days 0 and 14. Serum was collected from theimmunized mice as well as naïve mice (negative control; non-vaccineinjected) on Days 14 and 28. Five mice were tested per group.

An antibody response to each vaccine was determined using an IndirectEnzyme-Linked Immunosorbent Assay (ELISA) method that was designed forthe detection of mouse antibodies against SARS-CoV-2 spike proteins.Each microtiter plate (Coster 3369, EIA/RIA Plate) was coated with 0.1μg of S1 per well or 0.2 μg of S1/S2 per well; testing indicated thatuse of 0.1 μg of S1/S2 produced similar results. Sera from mice werediluted 100-fold in blocking buffer (0.5% Bovine albumin serum (BSA) in0.05% Polysorbate 20-20). The diluted sera were serially diluted induplicate to a final dilution of 6,400 times the initial sera. Theplates were incubated at 5° C. overnight (16-18 hours). After washingthe plates, horseradish peroxidase-conjugated Goat anti-mouse IgGsecondary Antibody (HRP), Sino Biological) was diluted (1 μL/10 mL) inblocking buffer and 100 ul was added to each well to detect theantibodies against spike protein. After a 1-2-hour incubation at 37° C.,the plates were washed and tetramethylbenzidine (TMB) substrate wasadded to detect Ab responses. The reaction was stopped afterapproximately 5 minutes with 1 N HCl. Immediately, absorbance wasmeasured at 450 nm using a Spectromax 190 microplate reader (MolecularDevices, CA). Endpoint titer for each mouse was determined as thehighest dilution of immune serum producing ELISA values (A₄₅₀ nm)greater than or equal to five times the binding detected with acorresponding dilution of naïve mice sera. The mean A₄₅₀ values obtainedfor the antibodies were calculated for each group of mice per vaccine.In all cases the results are the mean value of IgG titer absorbance forfive mice. Samples were stored at 5 C, 25 C, and 40 C up to one monthfor stability evaluation. Stability was assessed by measurement ofparticle size and UV absorbance. Freeze-dried vaccine was found to bestable for at least two weeks at 40 degrees C., and one month at 25degrees; as a result, vaccine may be suitable for transport and storagewithout refrigeration.

All samples were analyzed on a Precision Detector Dynamic LightScattering (DLS) instrument PD2000DLS^(plus) and PDDLS/CoolBatch 90Tusing quartz cuvettes (Precision Detectors). Liposomal samples werediluted 197 times in histidine sucrose buffer, from an original 25 mg/mlsuspension. Measurements were done at 20° C. using a refractive index of1.3479 and a viscosity of 0.0133 Poise for a 10% sucrose solution.Sample time was 15 μsec with a 3 sec run duration and a total of 60accumulations per measurement. Data was analyzed using PrecisionDeconvolve software. Stock solutions of S1 and S1S2 (at 250 and 550μg/mL, respectively) and also 10 μg/mL solutions of S1 and S1S2 werealso analyzed without dilution. Particle size was found to be stablebetween samples.

Referring now to FIG. 7, a bar graph illustrates experimental resultscomparing IgG immune response (vertical axis) measured from seraextracted from mice vaccinated using embodiments of disclosed vaccine inwhich the antigen was an S1 protein without S2 protein component. ELISAwas performed with S1-immobilized plates. Naïve samples (unvaccinated)were compared to sera samples from mice vaccinated with four otherformulations of a solution of S1 glycoproteins, denoted “S1,”, a vaccineformulated using a lipid blend that has not be lyophilized, which wasmixed with antigens (in this case S1), which process is referred to inthe graphs as “A-S1” as described above in reference to FIG. 6, aformulation of lipid blend lyophilized and reconstituted with S1 spikeproteins, referred to in the graphs as “B-S1” as described above inreference to FIG. 6, and a formulation of freeze-dried lipid blendreconstituted with spike protein (S1) solution, then freeze-dried again,and reconstituted a second time, denoted formulation “D-S1” as describedabove in reference to FIG. 6. As shown in FIG. 6, A-S1, B-S1, and D-S1all formulations significantly outperformed S1 alone and controlresulting in a statistically significant increase in S1-neutralizingantibody response, while B-S1 outperformed A-S1 and D-S1 by asignificant margin.

Referring now to FIG. 8, a bar graph illustrates experimental resultscomparing immune response (vertical axis) measured for sera from micevaccinated using embodiments of disclosed vaccine in which the antigenwas an S1 with no S2, placed on plates coated with S1S2. As before naïvecontrol, S1, A-S1, B-S1, and D-S1 solutions were used. As illustrated inFIG. 7, all three vaccines were immunogenic.

Referring now to FIG. 9, a bar graph illustrates experimental resultscomparing immune response (vertical axis) measured for blood from micevaccinated using embodiments of disclosed vaccine in which the antigenwas an S1S2 vaccine on a plate coated with S1. Formulations included“S1S2,” which was a solution of S1S2 alone, “A-S1S2,” prepared asdescribed above for A-S1, but with S1S2 spike proteins instead of S1alone, “B-S1S2,” prepared as described above for B-S1, but with S1S2spike proteins instead of S1 alone, and “D-S1S2,” prepared as describedabove for D-S1, but with S1S2 spike proteins instead of S1 alone. Allformulations were significantly more immunogenic than control, withB-S1S2 far outperforming others.

Referring now to FIG. 10, a bar graph illustrates experimental resultscomparing immune response (vertical axis) measured for blood from micevaccinated using embodiments of disclosed vaccine in which the antigenwas an S1S2 vaccine on a plate coated with S1S2. Formulations includedS1S2, A-S1S2, B-S1S2, and D-S1S2. All formulations were significantlymore immunogenic than control, with D-S1S2 and A-S1S2 outperformingB-S1S2.

Referring now to FIG. 11, a graph showing experimental results ofstability assessments at one month as described above is provided.Formulations C-S1 and C-S1S2 were not lyophilized. Formulations B-S1 andB-S1S2, which have been described above, were reconstituted, withantigens, as described above at a time of stability assessment.Formulations D-S1 and D-S1S2 were reconstituted at a time of assessmentas well. As illustrated in FIG. 11 and shown in Table 1 with respect toparticle size, Lyophilized vaccines (D-S1 and D-S1S2) and lyophilizedadjuvant (B) were stable at 25 C for at least 5 weeks, and at 40 C forat least 2 weeks. When lyophilized adjuvant B was reconstituted at t0with S1 protein, it resulted in a vaccine particle with a size that didnot change significantly if the adjuvant was stored for 5 weeks at 25 orfor 2 weeks at 40 C. This would indicate that the liposomal adjuvant Bis stable at 25 for at least 5 weeks and at 40 C for at least 2 weeks.FIGS. 12 and 13 show representative histograms for effect of temperatureon B and D formulations, respectively. Lyophilization increased vaccinestability at 25 C and 40 C. Liquid vaccines C-S1 and C-S1S2 were notstable at either temperature for 2 weeks.

TABLE 1 Vaccine t0 2 w 25 C. 5 w, 25 C. 2 w 40 C. 5 w, 40 C. B 176 ± 3173 ± 6  B-S1 176 ± 3 — 186 ± 6 170 ± 2  166 ± 3 B-S1S2 178 ± 2 — — — —C-S1 193 ± 4 225 ± 5 — 1,257 ± 266  — C-S1S2 196 ± 3 221 ± 3 — 326 ± 30— D-S1 198 ± 4 —  203 ± 16 326 ± 74 262 ± 9 D-S1S2 218 ± 7 209 ± 5 269 ±22  355 ± 52

The foregoing has been a detailed description of illustrativeembodiments of the invention. Various modifications and additions can bemade without departing from the spirit and scope of this invention.Features of each of the various embodiments described above may becombined with features of other described embodiments as appropriate inorder to provide a multiplicity of feature combinations in associatednew embodiments. Furthermore, while the foregoing describes a number ofseparate embodiments, what has been described herein is merelyillustrative of the application of the principles of the presentinvention. Additionally, although particular methods herein may beillustrated and/or described as being performed in a specific order, theordering is highly variable within ordinary skill to achieve embodimentsaccording to this disclosure. Accordingly, this description is meant tobe taken only by way of example, and not to otherwise limit the scope ofthis invention.

Exemplary embodiments have been disclosed above and illustrated in theaccompanying drawings. It will be understood by those skilled in the artthat various changes, omissions, and additions may be made to that whichis specifically disclosed herein without departing from the spirit andscope of the present invention.

1-11. (canceled)
 12. A method of manufacturing an immunogeniccomposition forming a vaccine, the method comprising: forming ananoparticle adjuvant, wherein: the adjuvant comprises at least ananoparticle, wherein the at least a nanoparticle comprises a lipidlayer exterior including at least one lipid, cholesterol, and a primaryalkyl amine including a positively charged amino group head and at leasta carbon tail; providing an antigen, the antigen comprising a spikeprotein from a coronavirus; and combining the antigen with thenanoparticle adjuvant, wherein combining the antigen with thenanoparticle adjuvant comprises: lyophilizing the nanoparticle adjuvant;combining the antigen with a buffer to form a suspension; andreconstituting the nanoparticle adjuvant with the suspension.
 13. Themethod of claim 12, wherein the lipid layer is positively charged. 14.The method of claim 12, wherein each nanoparticle includes a liposome.15. The method of claim 12, wherein the spike protein includes an S1protein.
 16. The method of claim 12, wherein the spike protein includesan S1S2 protein. 17-19. (canceled)
 20. The method of claim 12 furthercomprising adding at least one lyoprotectant to the combined adjuvantand antigen.
 21. The method of claim 12, wherein the plurality of spikeproteins comprises recombinant proteins.
 22. The method of claim 12,wherein the primary alkyl amine includes stearylamine.
 23. The method ofclaim 12, wherein the primary alkyl amine comprises a branched alkylamine.
 24. The method of claim 12, wherein the antigen has an electriccharge with a first polarity; and lipid layer exterior has an electriccharge with a second polarity, wherein the first polarity differs fromthe second polarity.
 25. The method of claim 12, wherein the antigen isabsorbed in the surface of the lipid layer.
 26. The method of claim 12,wherein the spike protein includes an S2 protein embedded in the lipidlayer and an S1 protein projecting from the lipid layer.
 27. The methodof claim 14, further comprising trapping the spike protein in an aqueouscompartment of the liposome.
 28. The method of claim 12, wherein thelipid layer exterior comprises non-phospholipids lipids.
 29. The methodof claim 27, wherein the non-phospholipids lipids are combined withpolyethylene glycols.
 30. The method of claim 12, wherein eachnanoparticle of the plurality of nanoparticles has a diameter between1000 and 2000 nanometers.
 31. The method of claim 12, wherein the lipidlayer further comprises a lipid bilayer, and wherein the spike proteinis entrapped within the lipid bilayer.
 32. The method of claim 12,wherein the lipid layer exterior includes cholesterol in an amountranging from 20 mol % to 30 mol %.
 33. The method of claim 12, whereinthe antigen include glycoprotein.